WO2024024427A1

WO2024024427A1 - Method for determining outer membrane detachment in cyanobacterium, device for determining outer membrane detachment in cyanobacterium, and program

Info

Publication number: WO2024024427A1
Application number: PCT/JP2023/024903
Authority: WO
Inventors: 征司児島
Original assignee: パナソニックＩｐマネジメント株式会社
Priority date: 2022-07-26
Filing date: 2023-07-05
Publication date: 2024-02-01

Abstract

The present disclosure provides a method for determining outer membrane detachment in a cyanobacterium that makes it possible to conveniently determine whether or not the outer membrane of the cyanobacterium has been detached from the cell wall. This method for determining outer membrane detachment in a cyanobacterium includes: a measurement step (S01) for measuring the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine and phenylalanine in the culture supernatant of the cyanobacterium; and a determination step (S02) for determining whether or not the outer membrane of the cyanobacterium has been detached from the cell wall on the basis of the concentration of the at least one amino acid measured in the measurement step.

Description

Method for determining outer membrane detachment of cyanobacteria, device for determining outer membrane detachment of cyanobacteria, and program

The present disclosure relates to a method for determining outer membrane detachment of cyanobacteria, an apparatus for determining outer membrane detachment of cyanobacteria, and a program.

Photosynthetic microorganisms such as cyanobacteria and algae are attracting attention as tools for realizing next-generation material production systems with low environmental impact. Substance production by photosynthetic microorganisms is carried out in an environment of normal temperature and pressure, using water and carbon dioxide (CO ₂ ) in the air with light as an energy source. Furthermore, with the recent development of genetic engineering technology, it has become possible to produce a wide range of chemical compounds using genetically modified photosynthetic microorganisms.Therefore, substance production using photosynthetic microorganisms is expected to be a next-generation technology that can achieve carbon neutrality. has been done.

As for the production of substances by such photosynthetic microorganisms, for example, techniques have been disclosed to improve the productivity of substances using genetically modified strains in which genes involved in the metabolism or biosynthesis of substances of photosynthetic microorganisms are modified. With this technology, for example, sucrose (Non-patent document 1), isobutanol (Non-patent document 2), fatty acids (Non-patent document 3), amino acids (Non-patent document 4), and proteins (Non-patent document 5), etc. Productivity is improved. Furthermore, for example, a technique for improving protein productivity using a genetically modified strain of cyanobacteria in which the outer membrane is peeled from the cell wall has also been disclosed (Patent Document 1).

International Publication No. 2021/100640

However, with the above-mentioned conventional technology, whether or not the state of the cells of the genetically modified strain of photosynthetic microorganisms is suitable for efficient substance production can be determined by, for example, electron microscopic observation of the cells or complicated biochemical methods. It is necessary to make a determination using an analytical method, which is time-consuming.

Therefore, the present disclosure provides a method for determining whether or not the outer membrane of cyanobacteria has peeled off from the cell wall, in which it is possible to easily determine whether or not the outer membrane has peeled off from the cell wall, as the state of the cyanobacteria cells is suitable for efficient substance production. Provided are a determination method, a determination device for outer membrane detachment of cyanobacteria, and a program.

A method for determining outer membrane detachment of cyanobacteria according to one aspect of the present disclosure includes measuring the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria. The method includes a measuring step, and a determining step of determining whether the outer membrane of the cyanobacterium has peeled off from the cell wall based on the concentration of the at least one amino acid measured in the measuring step.

In addition, the apparatus for determining outer membrane detachment of cyanobacteria according to one aspect of the present disclosure determines the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria. The method includes a measuring section that performs measurement, and a determining section that determines whether or not the outer membrane of the cyanobacteria is peeled from the cell wall based on the concentration of the at least one amino acid measured by the measuring section.

Further, the program according to one aspect of the present disclosure is configured to determine whether the cyanobacterium is out of the cyanobacteria based on the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of the cyanobacteria. This is a program that causes a computer to execute a method for determining whether or not a membrane has detached from a cell wall.

According to the method for determining outer membrane detachment of cyanobacteria, the apparatus for determining outer membrane detachment of cyanobacteria, and the program of the present disclosure, it is possible to easily determine whether the outer membrane of cyanobacteria has detached from the cell wall. Can be done.

FIG. 1 is a block diagram showing an example of the functional configuration of a cyanobacterial outer membrane exfoliation determination apparatus according to an embodiment. FIG. 2A is a flowchart illustrating an example of a flow of a method for determining outer membrane detachment of cyanobacteria according to an embodiment. FIG. 2B is a flowchart showing the detailed flow of step S02 in FIG. 2A. FIG. 3 is a diagram schematically showing the cell surface layer of cyanobacteria. FIG. 4 is a transmission electron microscopy image of an ultrathin section of the modified cyanobacteria of Example 1. FIG. 5 is an enlarged image of the broken line area A in FIG. FIG. 6 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 2. FIG. 7 is an enlarged image of the broken line area B in FIG. FIG. 8 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Comparative Example 1. FIG. 9 is an enlarged view of the broken line area C in FIG. FIG. 10 is a graph showing the amount of protein in the culture supernatants of the modified cyanobacteria of Example 1, Example 2, and Comparative Example 1 (n=3, error bar=SD). FIG. 11 is a diagram showing the results of amino acid analysis by LC-MS/MS of culture supernatants of two types of cyanobacteria. FIG. 12 shows SEQ ID NO: 1 to SEQ ID NO: 3. FIG. 13 shows SEQ ID NO: 4 to SEQ ID NO: 6. FIG. 14 shows SEQ ID NO:7. FIG. 15 shows SEQ ID NO:8. FIG. 16 shows SEQ ID NO:9. FIG. 17 shows SEQ ID NO: 10. FIG. 18 shows SEQ ID NO: 11. FIG. 19 shows SEQ ID NO: 12 to SEQ ID NO: 18. FIG. 20 shows SEQ ID NO: 19 to SEQ ID NO: 22.

(Findings that formed the basis of this disclosure)
Photosynthetic microorganisms such as cyanobacteria and algae are attracting attention as tools for realizing next-generation material production systems with low environmental impact. In particular, in the production of substances using cyanobacteria, there is a need for a method to efficiently and easily evaluate and manage cyanobacterial cells and the state of their culture.

Therefore, the present inventors focused on cyanobacteria as photosynthetic microorganisms used for substance production. Cyanobacteria (also called cyanobacteria or blue-green algae) are a group of eubacteria that decompose water through photosynthesis to produce oxygen, and use the energy obtained to fix _CO2 in the air. Note that, depending on the species, cyanobacteria can also fix nitrogen (N ₂ ) in the air. In addition, cyanobacteria are known to grow quickly and have high light utilization efficiency, and in addition, they are easier to genetically manipulate than other algae species. Active research and development is underway. As mentioned above, examples of substance production by cyanobacteria (more specifically, modified cyanobacteria) include sucrose (Non-Patent Document 1), isobutanol (Non-Patent Document 2), fatty acids (Non-Patent Document 3), Production of amino acids (Non-Patent Document 4), proteins (Non-Patent Document 5), etc. has been reported.

For example, Non-Patent Document 1 discloses that a genetically modified strain of Synechococcus elongatus in which genes involved in the sucrose biosynthesis pathway have been modified has improved sucrose productivity compared to the wild strain.

For example, Non-Patent Document 2 describes the production of isobutanol using a genetically modified strain that overexpresses ribulose-1,5-bisphosphate carboxylase/oxidase (Rubisco), which was created by genetically engineering Synechococcus elongatus PCC7942. It has been disclosed that the sex of the strain was improved over that of the wild strain.

For example, Non-Patent Document 3 discloses that a genetically modified strain of Synechocystis sp. PCC6803 in which an acyl-acyl transport protein thioesterase gene has been introduced has improved fatty acid productivity compared to the wild strain.

In addition, Non-Patent Document 4 describes that the tryptophan productivity of a tryptophan overproducing strain isolated by subjecting the wild strain of Synechocystis sp. PCC 6803 to random mutagenesis and selection using amino acids itself It has been disclosed that this has been improved.

In addition, in Non-Patent Document 5, the chloroplast of Chlamydomonas reinhardtii, a type of algae, was genetically engineered to produce a chimeric protein consisting of a 25 kDa Plasmodium falciparum surface protein (Pfs25) fused to the β subunit of cholera toxin (CtxB). It has been disclosed that genetically modified algae that produce (CtxB-Pfs25) in cells can be used as an oral vaccine for malaria.

However, in the above-mentioned conventional technology, even if the target substance is produced within the cells of the genetically modified strain of the photosynthetic microorganism, it accumulates without being secreted outside the cell, or it is difficult to secrete it effectively outside the cell. It is necessary to collect the desired substance by crushing the cells, which takes time and effort to produce the substance. Furthermore, since various substances exist within cells, it may be necessary to remove these substances and purify the target substance, which lowers the recovery rate of the target substance. Furthermore, each time a target substance is produced, it is necessary to prepare a new genetically modified strain, which is time-consuming and increases production costs. As described above, in the conventional techniques described above, the production efficiency of substances by photosynthetic microorganisms is still at a low level, and there is a desire to develop a technique with higher production efficiency.

The structure of the cell wall and cell membrane of cyanobacteria has low permeability to substances produced within the cell, and it is not easy to artificially modify the structure of the cell membrane and cell wall to improve the ability to secrete and produce substances. In particular, substances with large molecular weights such as proteins (also referred to as high molecular weight compounds) are difficult to secrete outside cells, unlike substances with relatively small molecular weights such as amino acids (also referred to as low molecular weight compounds).

However, it has been reported that deletion of the slr1841 gene or slr0688 gene, which is involved in adhesion between the outer membrane and cell wall of cyanobacteria and contributes to the structural stability of the cell surface, results in loss of the growth ability of cyanobacterial cells. (Non-Patent Document 6: Hikaru Kobata, Studies on molecular basis of cyanobacterial outer membrane function and its evolutionary relationship with primitive chloroplasts, PhD thesis, [Online], 2018.03.27, Internet: <URL: http://hdl .handle.net/10097/00122689>, and Non-Patent Document 7: Seiji Kojima, Elucidation and application of membrane stabilization and substance permeation mechanisms derived from bacteria that function in the chloroplast surface membrane, Grants-in-Aid for Scientific Research, [Online] , 2018.04.23, Internet: <URL: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18H02117>).

As a technique for solving the above problem, Patent Document 1 describes a modified cyanobacterium in which the function of a protein involved in binding between the outer membrane of cyanobacteria and the cell wall (hereinafter also referred to as binding-related protein) is suppressed or lost, and A method for producing proteins using modified cyanobacteria is disclosed. In the technology described in Patent Document 1, the outer membrane of the cyanobacterium is peeled off from the cell wall while the modified cyanobacterium maintains its ability to proliferate. The resulting protein is secreted to the outside of the cell, allowing for efficient protein production.

However, in the above-mentioned conventional technology, it is difficult to determine whether or not the cell state of modified cyanobacteria is suitable for efficient substance production by observing cells with an electron microscope or by using complicated biochemical analysis methods. It is time-consuming because it requires a determination.

In addition, it is known that the productivity of substances improves when the outer membrane of cyanobacteria is detached from the cell wall, but whether or not the outer membrane is detached requires electron microscopy and complicated biochemical analysis techniques. It is necessary to make a determination using , and no simple method has been developed at this time.

Therefore, the present inventors have intensively investigated a method for easily determining whether or not the outer membrane has peeled off from the cell wall, assuming that the state of the cyanobacterial cells is suitable for efficient substance production. As a result, they found that it is possible to easily determine whether the outer membrane of cyanobacteria has detached from the cell wall based on the concentration of a specific amino acid in the culture supernatant of cyanobacteria. Therefore, according to the present disclosure, it is possible to determine whether or not cyanobacteria are suitable for efficient substance production, and thus it is possible to improve the productivity of substances by cyanobacteria.

(Summary of this disclosure)
An overview of one aspect of the present disclosure is as follows.

Accordingly, the method for determining outer membrane detachment of cyanobacteria involves collecting the culture supernatant of cyanobacteria and measuring the concentration of at least one amino acid selected from the group consisting of the above four amino acids in the culture supernatant. Therefore, peeling of the outer membrane of cyanobacteria can be easily determined.

For example, in the method for determining outer membrane detachment of cyanobacteria according to one aspect of the present disclosure, in the measuring step, the concentration of four amino acids, isoleucine, leucine, tyrosine, and phenylalanine, is measured; Based on the concentrations of the four amino acids, it may be determined whether the outer membrane of the cyanobacterium is detached from the cell wall.

As a result, the method for determining outer membrane detachment of cyanobacteria measures the concentration of the above four amino acids in the culture supernatant of cyanobacteria, and determines outer membrane detachment of cyanobacteria based on the concentration of these amino acids. Therefore, outer membrane detachment of cyanobacteria can be determined with higher accuracy.

For example, in the method for determining outer membrane detachment of cyanobacteria according to one aspect of the present disclosure, in the determination step, if at least one of the concentrations of the at least one amino acid is equal to or higher than a threshold value, the outer membrane of the cyanobacteria is removed. If it is determined that the outer membrane of the cyanobacterium has detached from the cell wall, and all of the concentrations of the at least one amino acid are below the threshold, it may be determined that the outer membrane of the cyanobacterium has not detached from the cell wall.

As a result, the method for determining outer membrane detachment of cyanobacteria is that if the concentration of even one of the at least one amino acid measured exceeds a threshold value, it is determined that the outer membrane of cyanobacteria has detached. Even if fluctuations occur due to the culture conditions, it is possible to accurately determine whether the outer membrane of cyanobacteria has peeled off.

For example, in the method for determining outer membrane detachment of cyanobacteria according to one aspect of the present disclosure, the threshold value may be 100 nM.

Accordingly, the method for determining outer membrane detachment of cyanobacteria is based on the determination of the concentration of at least one amino acid selected from the group consisting of the above four amino acids contained in the culture supernatant of cyanobacteria whose outer membrane has detached from the cell wall. Detachment of the outer membrane of cyanobacteria can be easily and quantitatively determined by whether or not it exceeds 100 nM.

As a result, the apparatus for determining cyanobacterial outer membrane detachment only needs to measure the concentration of at least one amino acid selected from the group consisting of the above four amino acids in the cyanobacterial culture supernatant. Adventitial detachment can be easily determined.

Thereby, the program determines whether or not the outer membrane of cyanobacteria has detached from the cell wall, based on the concentration of at least one amino acid selected from the group consisting of the above four amino acids in the culture supernatant of cyanobacteria. Since the computer determines this, the computer can easily determine outer membrane detachment of cyanobacteria.

Hereinafter, embodiments will be specifically described with reference to the drawings.

Note that all embodiments described below are comprehensive or specific examples. The numerical values, materials, steps, order of steps, etc. shown in the following embodiments are merely examples, and do not limit the present disclosure. Further, among the constituent elements in the following embodiments, constituent elements that are not described in the independent claims indicating the most significant concept will be described as arbitrary constituent elements.

Furthermore, each figure is not necessarily strictly illustrated. In each figure, substantially the same configurations are denoted by the same reference numerals, and overlapping explanations may be omitted or simplified.

In addition, in the following, the numerical range does not represent only a strict meaning, but includes a substantially equivalent range, for example, measuring the amount of protein (for example, number or concentration, etc.) or the range thereof.

Furthermore, in this specification, both a bacterial body and a cell represent one individual cyanobacterium.

(Embodiment)
[1. Definition]
In this specification, the identity of base sequences and amino acid sequences is calculated by the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it was calculated by performing pairwise analysis using the BLAST program available on the website of NCBI (National Center for Biotechnology Information) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Ru. Information regarding cyanobacterial genes and proteins encoded by the genes is published, for example, in the NCBI database mentioned above and Cyanobase (http://genome.microbedb.jp/cyanobase/). From these databases, the amino acid sequences of proteins of interest and the base sequences of genes encoding those proteins can be obtained.

[2. Device for determining outer membrane detachment of cyanobacteria]
Next, a cyanobacterial outer membrane exfoliation determination device (hereinafter also simply referred to as a determination device) according to the present embodiment will be described. FIG. 1 is a block diagram showing an example of the functional configuration of a cyanobacterial outer membrane exfoliation determination apparatus according to the present embodiment.

As shown in FIG. 1, the determination device 100 includes, for example, a measurement section 110, a control section 120, a storage section 130, an input reception section 140, and a display section 150. The control unit 120 includes, for example, a determination unit 122.

The measurement unit 110 measures, for example, the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria. The measurement unit 110 only needs to be able to measure the concentration of amino acids in the culture supernatant, and may be a conventionally known amino acid analyzer. The measurement unit 110 is, for example, a liquid chromatography (HPLC), a liquid chromatograph mass spectrometer (LC/MS), a liquid chromatograph tandem mass spectrometer (LC-MS/MS), or the like.

Although not shown, the measurement unit 110 includes, for example, a control unit that controls the operation of the measurement unit 110, and the control unit controls the measurement unit 110 according to a control signal output from the control unit 120 of the determination device 100. control the behavior of In the example of FIG. 1, the determination device 100 includes the measurement unit 110, but may not include the measurement unit 110. In this case, the measurement unit 110 is a measurement device, and the determination device 100 is connected to the measurement device via communication.

The control unit 120 performs information processing to control the operation of the determination device 100. The control unit 120 is implemented, for example, by a microcomputer, but may also be implemented by a processor or a dedicated circuit. Specifically, the control unit 120 includes a determination unit 122. The determination unit 122 is realized by a processor executing a program for performing the above information processing.

Based on the concentration of at least one amino acid measured by the measurement unit 110, the determination unit 122 determines whether the outer membrane of the cyanobacteria has peeled off from the cell wall. For example, the function of the determination unit 122 is realized by a CPU (Central Processing Unit) executing a program stored in the storage unit 130. The specific function of the determination unit 122 will be explained in the next section.

The storage unit 130 is a storage device in which control programs executed by the control unit 120 and the like are stored. The storage unit 130 is realized by, for example, a semiconductor memory.

The input accepting unit 140 accepts user operation input. Specifically, the input reception unit 140 is realized by a mouse, a microphone, a touch panel, or the like.

The display unit 150 is a display device that displays information to be presented to the user based on the control of the control unit 120. The display unit 150 is realized by a liquid crystal panel or an organic EL (Electro Luminescence) panel.

[3. Method for determining outer membrane detachment of cyanobacteria]
Next, a method for determining outer membrane detachment of cyanobacteria according to the present embodiment will be described with reference to FIGS. 2A and 2B. FIG. 2A is a flowchart illustrating an example of a flow of a method for determining outer membrane detachment of cyanobacteria according to an embodiment. FIG. 2B is a flowchart showing the detailed flow of step S02 in FIG. 2A.

The determination method is implemented by the determination device described above. For example, as shown in FIG. 2A, the measurement section of the determination device detects at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria introduced into the measurement section. Measure the concentration (S01). The culture supernatant may be sampled at predetermined intervals (for example, on a daily basis) after the start of the main culture. Sampling may be performed manually by a user or may be performed automatically. Isoleucine, leucine, tyrosine, and phenylalanine are characteristically secreted into the culture supernatant when cyanobacterial cells from which the outer membrane has been peeled are cultured.

Next, the determination unit of the determination device determines whether the outer membrane of the cyanobacteria has peeled off from the cell wall, based on the concentration of at least one amino acid measured by the measurement unit (S02). More specifically, as shown in FIG. 2B, in step S02, the determination unit determines whether all of the concentrations of at least one amino acid measured by the measurement unit are less than a threshold (S11). If it is determined that all of the measured concentrations of at least one amino acid are less than the threshold value (Yes in S11), it is determined that the outer membrane of the cyanobacteria has not peeled off from the cell wall (S12).

On the other hand, if at least one of the concentrations of the at least one amino acid measured by the measurement unit is equal to or higher than the threshold (No in S11), the determination unit determines that the outer membrane of the cyanobacteria has detached from the cell wall ( S13).

In addition, in the method for determining outer membrane detachment of cyanobacteria, the outer membrane of cyanobacteria is determined by measuring (in other words, quantifying) the concentrations of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria. However, the amino acids and biomolecules (for example, intracellular metabolites) to be measured are not limited to these.

Note that the measurement method executed in step S01 may be any method that can measure amino acids or biomolecules, such as liquid chromatography (HPLC), liquid chromatograph mass spectrometer (LC/MS), or liquid chromatography. For example, the measurement method performed in step S01 may be an enzyme assay for detecting a specific amino acid or biomolecule. It may be.

[4. cyanobacteria]
Cyanobacteria, also called blue-green algae or cyanobacteria, are a group of prokaryotes that perform photosynthesis while collecting light energy with chlorophyll and electrolyzing water to generate oxygen. Cyanobacteria are highly diverse, and include, for example, unicellular species such as Synechocystis sp. PCC 6803 and filamentous multicellular species such as Anabaena sp. PCC 7120. Regarding the growing environment, there are thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. In addition, species such as Microcystis aeruginosa, which has gas vesicles and produces toxins, and Gloeobacter violaceus, which does not have thylakoid but has a protein called phycobilisome, which is a light-harvesting antenna on its plasma membrane, have unique characteristics. There are many species that can be mentioned.

FIG. 3 is a diagram schematically showing the cell surface layer of cyanobacteria. As shown in Figure 3, the cell surface layer of cyanobacteria is composed of, in order from the inside, a plasma membrane (also called inner membrane 1), peptidoglycan 2, and outer membrane 5, which is a lipid membrane that forms the outermost layer of the cell. Ru. Sugar chains 3 composed of glucosamine, mannosamine, etc. are covalently bonded to peptidoglycan 2, and pyruvate is bonded to these covalently bonded sugar chains 3 (Non-Patent Document 8: Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, the peptidoglycan 2 and the covalent sugar chain 3 are collectively referred to as a cell wall 4. In addition, the gap between the plasma membrane (that is, the inner membrane 1) and the outer membrane 5 is called the periplasm, and is used for protein decomposition or three-dimensional structure formation, lipid or nucleic acid decomposition, or the uptake of extracellular nutrients. There are various enzymes involved.

The SLH domain-retaining outer membrane protein (for example, Slr1841 in the figure) consists of a C-terminal region embedded in the lipid membrane (also referred to as outer membrane 5) and an N-terminal SLH domain 7 that protrudes from the lipid membrane. It is widely distributed in cyanobacteria and bacteria belonging to the class Negativicutes, a group of Gram-negative bacteria (Non-Patent Document 9: Kojima et al., 2016, Biosci. Biotech. Biochem., 10:1954-1959). The region embedded in the lipid membrane (i.e. outer membrane 5) forms a channel to allow the passage of hydrophilic substances through the outer membrane, while the SLH domain 7 has the function of binding to the cell wall 4 (non-transparent membrane 5). Patent Document 10: Kowata et al., 2017, J. Bacteriol., 199:e00371-17). In order for SLH domain 7 to bind to cell wall 4, covalent sugar chain 3 in peptidoglycan 2 needs to be modified with pyruvate (Non-Patent Document 11: Kojima et al., 2016, J. Biol Chem., 291:20198-20209). Examples of genes encoding SLH domain-retaining outer membrane protein 6 include slr1841 or slr1908 held by Synechocystis sp. PCC 6803, or oprB held by Anabaena sp. 90.

The enzyme that catalyzes the pyruvate modification reaction of covalent sugar chain 3 in peptidoglycan 2 (hereinafter referred to as cell wall-pyruvate modification enzyme 9) was identified in the Gram-positive bacterium Bacillus anthracis and named CsaB. (Non-patent document 12: Mesnage et al., 2000, EMBO J., 19:4473-4484). Among cyanobacteria whose genome sequences have been published, many species possess genes encoding homologous proteins with amino acid sequence identity of 30% or more with CsaB. Examples include slr0688 held by Synechocystis sp. PCC 6803 or synpcc7502_03092 held by Synechococcus sp. 7502.

In cyanobacteria, _CO2 fixed through photosynthesis is converted into various amino acids through multi-step enzymatic reactions. Using these as raw materials, proteins are synthesized within the cytoplasm of cyanobacteria. Some of these proteins function within the cytoplasm, while others are transported from the cytoplasm to the periplasm and function within the periplasm. However, no case of active secretion of proteins outside the cell has been reported in cyanobacteria to date.

Because cyanobacteria have a high photosynthetic ability, they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria have very few channel proteins in their outer membrane 5 that allow organic substances to pass therethrough, such as the organic substance channel protein 8 (eg, Slr1270) in FIG. For example, in Synechocystis sp. PCC 6803, organic channel protein 8, which allows organic matter to pass through, is present in only about 4% of the total protein content of outer membrane 5. On the other hand, in order for cyanobacteria to take in inorganic ions necessary for growth into cells with high efficiency, only inorganic ions can pass through, as shown in SLH domain-retaining outer membrane protein 6 (e.g., Slr1841) shown in Figure 3. The outer membrane 5 contains many ion channel proteins that cause For example, in Synechocystis sp. PCC 6803, ion channel proteins that permeate inorganic ions account for about 80% of the total protein content of the outer membrane 5.

In this way, cyanobacteria have very few channels in the outer membrane 5 that allow organic substances such as proteins to pass through, so it is thought that it is difficult to actively secrete proteins produced within the bacterium to the outside of the bacterium. There is. Furthermore, although the structure of the cell wall and cell membrane of cyanobacteria influences protein permeability, it is not easy to artificially modify the cell membrane and cell wall structure to improve protein secretion production ability. For example, Non-Patent Document 6 and Non-Patent Document 7 state that when the slr1841 gene or slr0688 gene, which is involved in adhesion between the outer membrane and the cell wall and contributes to the structural stability of the cell surface, is deleted, the proliferation ability of the cell is reduced. It is stated that it will be lost.

[5. Modified cyanobacteria]
Next, the cyanobacteria (hereinafter referred to as modified cyanobacteria) according to the present embodiment will be described with reference to FIG. 3. Further, below, proteins will be explained as an example of substances produced using modified cyanobacteria.

In the modified cyanobacteria according to this embodiment, the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 (so-called binding-related protein) is suppressed or lost. More specifically, for example, the modified cyanobacteria is such that the total amount of proteins involved in binding between the outer membrane 5 and the cell wall 4 (i.e., binding-related proteins) is lower than that in the parent strain (i.e., parent cyanobacteria). The total amount of protein is suppressed to 30% or more and 70% or less. Here, for example, "the total amount of binding-related proteins is suppressed to 30% of the total amount of the protein in the parent strain" means that 70% of the total amount of the protein in the parent strain has been lost and 30% remains. It means the state of being. As a result, in the modified cyanobacteria, the bond between the outer membrane 5 and the cell wall 4 (for example, the amount of bond and the binding force) is partially reduced, so that the outer membrane 5 is easily partially detached from the cell wall 4. Therefore, the modified cyanobacteria has improved protein secretion productivity, which secretes proteins produced within the bacterial cells to the outside of the bacterial cells. Furthermore, since it is not necessary to crush the bacterial cells to recover proteins, the modified cyanobacteria can be repeatedly used to produce proteins even after the proteins are recovered. In this specification, the production of proteins by modified cyanobacteria within the bacterial cells is referred to as production, and the secretion of the produced proteins to the outside of the bacterial cells is referred to as secretory production.

The protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9. In the present embodiment, in the modified cyanobacteria, the function of at least one protein, for example, the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9, is suppressed. For example, in the modified cyanobacteria, (i) at least one function of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 may be suppressed, and (ii) the SLH domain-retaining type that binds to the cell wall 4 may be suppressed. At least one of the expression of the outer membrane protein 6 and the expression of an enzyme that catalyzes the pyruvate modification reaction of sugar chains bound to the surface of the cell wall 4 (ie, the cell wall-pyruvate modifying enzyme 9) may be suppressed. This reduces the bond (that is, the bond amount and binding force) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent sugar chain 3 on the surface of the cell wall 4. Therefore, the outer membrane 5 easily detaches from the cell wall 4 in areas where these bonds are weakened. By partially detaching the outer membrane 5 from the cell wall 4, proteins present in the cells of the modified cyanobacteria, particularly in the periplasm, tend to leak out of the cells (outside the outer membrane 5).

As mentioned above, cyanobacteria have a high photosynthetic ability, so they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria only need light, air, water, and a small amount of inorganic substances to cultivate cyanobacteria, and cyanobacteria take inorganic substances into cells through ion channels in the outer membrane 5 and produce proteins within the cells. In particular, various proteins are present in the periplasm, which is the space between the outer membrane 5 and the cell wall 4. In the modified cyanobacterium according to the present embodiment, the functions of proteins involved in binding between the outer membrane 5 and the cell wall 4 are suppressed. As a result, the outer membrane 5 is easily partially peeled off from the cell wall 4. Then, proteins within the periplasm leak into the culture solution from the peeled portion of the outer membrane 5. As a result, the modified cyanobacteria improves the productivity of protein secretion, which secretes proteins produced within the bacterial cells to the outside of the bacterial cells.

Hereinafter, the outer membrane 5 is modified to partially detach from the cell wall 4 by suppressing the function of at least one binding-related protein of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9. The following cyanobacteria will be explained in more detail.

The cyanobacterium that is the parent microorganism of the modified cyanobacterium according to the present embodiment, which has not yet suppressed at least one of the expression of the SLH domain-retaining outer membrane protein 6 and the expression of the cell wall-pyruvate modifying enzyme 9 The type of the "parent strain" or "parent cyanobacteria") is not particularly limited and may be any type of cyanobacteria. For example, the parent cyanobacteria may be of the genus Synechocystis, Synechococcus, Anabaena, or Thermosynechococcus, among which Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1. Good too. The parent strain may be a wild cyanobacterium, as long as the total amount of binding-related proteins has not been suppressed to 30% or more and 70% or less, or it may be a modified strain that is equivalent to the wild one. binding-related proteins.

The amino acid sequences of the SLH domain-retaining outer membrane protein 6 and the enzyme that catalyzes the cell wall-pyruvate modification reaction (i.e., the cell wall-pyruvate modification enzyme 9) in these parent cyanobacteria, and the genes encoding their binding-related proteins. The base sequence and the position of the gene on the chromosomal DNA or plasmid can be confirmed in the NCBI database and Cyanobase mentioned above.

Note that the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvic acid modifying enzyme 9, whose functions are suppressed in the modified cyanobacteria according to the present embodiment, can be used in any parent cyanobacteria as long as they are possessed by the parent cyanobacteria. They are not limited by the location of the genes encoding them (for example, on chromosomal DNA or on plasmids).

For example, the SLH domain-retaining outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042 when the parent cyanobacterium belongs to the genus Synechocystis, or may be NIES970_09470 when the parent cyanobacterium belongs to the genus Synechococcus. Often, when the parent cyanobacteria is of the genus Anabaena, it may be Anacy_5815 or Anacy_3458, etc. When the parent cyanobacteria is of the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc. When the parent cyanobacteria is of the genus Cyanothece, it may be A0A3B8XX12_9CYAN, etc. When the parent cyanobacterium belongs to the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. When the parent cyanobacterium belongs to the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, and when the parent cyanobacterium belongs to the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. When the parent cyanobacterium belongs to the genus Crocosphaera, it may be B1WRN6_CROS5, and when the parent cyanobacterium belongs to the genus Pleurocapsa, it may be K9TAE4_9CYAN.

More specifically, the SLH domain-retaining outer membrane protein 6 is, for example, Slr1841 (SEQ ID NO: 1) of Synechocystis sp. PCC 6803, NIES970_09470 (SEQ ID NO: 2) of Synechococcus sp. NIES-970, or Anabaena cylindrica PCC. Anacy_3458 (SEQ ID NO: 3) of 7122 may be used. Alternatively, it may be a protein that has an amino acid sequence that is 50% or more identical to these SLH domain-retaining outer membrane proteins 6.

As a result, in the modified cyanobacteria, for example, (i) any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 above or any of these SLH domain-retaining outer membrane proteins 6 and amino acids; The function of the protein whose sequence is 50% or more identical may be suppressed, and (ii) any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 above or any of these SLHs. Expression of a protein whose amino acid sequence is 50% or more identical to domain-retaining outer membrane protein 6 may be suppressed. Therefore, in the modified cyanobacteria, (i) the function of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 or a protein having a function equivalent to the SLH domain-retaining outer membrane protein 6 is suppressed, or (ii) ) The expression level of the SLH domain-retaining outer membrane protein 6 or a protein having the same function as the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the binding domain (for example, SLH domain 7) for binding the outer membrane 5 to the cell wall 4 has a reduced binding amount and binding force with the cell wall 4, so The membrane 5 is easily partially detached from the cell wall 4.

Generally, it is said that if the amino acid sequences of a protein are 30% or more identical, the protein has a high degree of homology in its three-dimensional structure and is therefore likely to have the same function as the protein in question. Therefore, the SLH domain-retaining outer membrane protein 6 whose function is suppressed is, for example, one of the amino acid sequences of the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 1 to 3 above, and 40% or more of the amino acid sequence, Preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and the cell wall 4 is shared. It may be a protein or a polypeptide that has the function of binding to a bound sugar chain 3.

Furthermore, for example, the cell wall-pyruvate modifying enzyme 9 may be Slr0688, etc. when the parent cyanobacterium belongs to the genus Synechocystis, and when the parent cyanobacterium belongs to the genus Synechococcus, it may be Syn7502_03092 or Synpcc7942_1529, etc. If the cyanobacterium belongs to the genus Anabaena, it may be ANA_C20348 or Anacy_1623, and if the parent cyanobacterium belongs to the genus Microcystis, it may be CsaB (NCBI access ID: TRU80220), or if the parent cyanobacterium belongs to the genus Cyanothece. If the parent cyanobacterium belongs to the genus Spirulina, it may be CsaB (NCBI access ID: WP_026079530.1), etc. If the parent cyanobacterium belongs to the genus Calothrix, it may be CsaB (NCBI access ID: WP_096658142.1), etc., and if the parent cyanobacterium belongs to the genus Nostoc, it may be CsaB (NCBI access ID: WP_099068528.1), etc. , if the parent cyanobacteria belongs to the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1), and if the parent cyanobacterium belongs to the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735), etc. Good too.

More specifically, the cell wall-pyruvate modifying enzyme 9 is, for example, Slr0688 (SEQ ID NO: 4) of Synechocystis sp. PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) of Synechococcus sp. Anacy_1623 (SEQ ID NO: 6) or the like may be used. Further, it may be a protein having an amino acid sequence that is 50% or more identical to these cell wall-pyruvate modifying enzymes 9.

As a result, in the modified cyanobacteria, for example, (i) any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above or any of these cell wall-pyruvate modifying enzymes 9 and the amino acid sequence The function of a protein that is 50% or more identical is suppressed, or (ii) any of the cell wall-pyruvic acid modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above or any of these cell wall-pyruvic acid Expression of a protein whose amino acid sequence is 50% or more identical to modified enzyme 9 is suppressed. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvate modifying enzyme 9 or a protein having an equivalent function to the enzyme is suppressed, or (ii) the function of the cell wall-pyruvate modifying enzyme 9 or the enzyme is suppressed. The expression level of proteins with equivalent functions decreases. This makes it difficult for the covalent sugar chains 3 on the surface of the cell wall 4 to be modified with pyruvate, so that the sugar chains 3 on the cell wall 4 interact with the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding strength are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are difficult to be modified with pyruvate, so the binding force between the cell wall 4 and the outer membrane 5 is weakened, and the outer membrane 5 becomes easily partially detached from the cell wall 4.

Additionally, as mentioned above, if the amino acid sequences of a protein are 30% or more identical, it is said that the protein is likely to have the same function as the protein. Therefore, the cell wall-pyruvate modifying enzyme 9 whose function is suppressed is, for example, an amino acid sequence of any one of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above, and 40% or more, preferably It consists of an amino acid sequence having an identity of 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and It may be a protein or polypeptide that has the function of catalyzing a reaction that modifies covalent sugar chain 3 with pyruvate.

In this specification, suppressing the function of the SLH domain-retaining outer membrane protein 6 refers to suppressing the ability of the protein to bind to the cell wall 4, or suppressing or losing the transport of the protein to the outer membrane 5. or suppress the ability of the protein to become embedded in the outer membrane 5 and function.

Note that suppressing the function of the cell wall-pyruvic acid modifying enzyme 9 means suppressing the function of the protein to modify the covalent sugar chain 3 of the cell wall 4 with pyruvate.

The means for suppressing the functions of these proteins is not particularly limited as long as it is a means normally used for suppressing the functions of proteins. The means includes, for example, deleting or inactivating the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, The method may include inhibiting the translation of transcription products of these genes, or administering an inhibitor that specifically inhibits these proteins.

In the present embodiment, the modified cyanobacteria may have a gene that expresses a protein involved in binding the outer membrane 5 and the cell wall 4 deleted or inactivated. As a result, in the modified cyanobacteria, the expression of the protein involved in the bond between the cell wall 4 and the outer membrane 5 is suppressed, or the function of the protein is suppressed, so that the bond between the cell wall 4 and the outer membrane 5 is suppressed. (so-called bond amount and bond strength) are partially reduced. As a result, in the modified cyanobacteria, the outer membrane 5 tends to partially detach from the cell wall 4, so that proteins produced within the bacterial body tend to leak out of the outer membrane 5, that is, to the outside of the bacterial body. Therefore, the modified cyanobacteria according to the present embodiment has improved protein secretion productivity for secreting proteins produced within the bacterial cells to the outside of the bacterial cells. Furthermore, since it is not necessary to crush the bacterial cells to recover proteins, the modified cyanobacteria can be repeatedly used to produce proteins even after the proteins are recovered.

The gene that expresses the protein involved in the binding between the outer membrane 5 and the cell wall 4 is, for example, at least one of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. There may be. In the modified cyanobacteria, at least one of the genes encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9 has been deleted or inactivated. Therefore, in the modified cyanobacteria, for example, (i) the expression of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 is suppressed, or (ii) the SLH domain-retaining outer membrane protein At least one function of cell wall-pyruvate modifying enzyme 6 and cell wall-pyruvate modifying enzyme 9 is suppressed. Therefore, the bond between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent sugar chain 3 on the surface of the cell wall 4 (that is, the bond amount and binding force) is reduced. This makes it easier for the outer membrane 5 to detach from the cell wall 4 at the portion where the bond between the outer membrane 5 and the cell wall 4 is weakened. Therefore, according to the modified cyanobacterium according to the present embodiment, the binding between the outer membrane 5 and the cell wall 4 is reduced, making it easier for the outer membrane 5 to partially detach from the cell wall 4. This makes it easier for protein to leak out of the bacterial body.

In this embodiment, in order to suppress at least one function of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 in cyanobacteria, for example, a gene encoding the SLH domain-retaining outer membrane protein 6 is used. and transcription of at least one gene encoding cell wall-pyruvate modifying enzyme 9 may be suppressed.

For example, the gene encoding SLH domain-retaining outer membrane protein 6 may be slr1841, slr1908, or slr0042 when the parent cyanobacterium belongs to the genus Synechocystis, and may be nies970_09470 when the parent cyanobacterium belongs to the genus Synechococcus. Often, when the parent cyanobacteria is of the genus Anabaena, it may be anacy_5815 or anacy_3458, etc. When the parent cyanobacteria is of the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc. When the parent cyanobacteria is of the genus Cyanothece, it may be A0A3B8XX12_9CYAN, etc. If the parent cyanobacteria belongs to the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. If the parent cyanobacterium belongs to the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, and if the parent cyanobacterium belongs to the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. When the parent cyanobacterium belongs to the genus Crocosphaera, it may be B1WRN6_CROS5, and when the parent cyanobacterium belongs to the genus Pleurocapsa, it may be K9TAE4_9CYAN. The base sequences of these genes can be obtained from the NCBI database or Cyanobase mentioned above.

More specifically, the genes encoding SLH domain-retaining outer membrane protein 6 include slr1841 (SEQ ID NO: 7) of Synechocystis sp. PCC 6803, nies970_09470 (SEQ ID NO: 8) of Synechococcus sp. NIES-970, and Anabaena cylindrica PCC. 7122 anacy_3458 (SEQ ID NO: 9), or a gene whose base sequence is 50% or more identical to these genes.

As a result, in the modified cyanobacteria, the gene encoding any of the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 7 to 9 above, or the base sequence of any of these genes, is 50% or more identical. Genes are deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above-mentioned SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to any of these proteins is suppressed, or (ii) the above-mentioned The function of any of the SLH domain-retaining outer membrane proteins 6 or a protein having a function equivalent to any of these proteins is suppressed. Therefore, in the modified cyanobacteria according to the present embodiment, the binding amount and binding force of the binding domain (for example, SLH domain 7) for binding the outer membrane 5 to the cell wall 4 with the cell wall 4 are reduced, 5 becomes easily partially detached from the cell wall 4.

As mentioned above, if the amino acid sequences of a protein are 30% or more identical, it is said that the protein is likely to have the same function as the protein. Therefore, if the base sequences of genes encoding proteins are 30% or more identical, it is considered that there is a high possibility that a protein having the same function as the protein will be expressed. Therefore, as a gene encoding the SLH domain-retaining outer membrane protein 6 whose function is suppressed, for example, any of the genes encoding the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 7 to 9 above can be used. From a base sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, still more preferably 90% or more with the base sequence. It may also be a gene that encodes a protein or polypeptide that has the function of binding to the covalent sugar chain 3 of the cell wall 4.

Furthermore, for example, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688, etc. when the parent cyanobacterium belongs to the genus Synechocystis, and may be syn7502_03092 or synpcc7942_1529, etc. when the parent cyanobacterium belongs to the genus Synechococcus. If the parent cyanobacterium belongs to the genus Anabaena, it may be ana_C20348 or anacy_1623, and if the parent cyanobacterium belongs to the genus Microcystis, it may be csaB (NCBI access ID: TRU80220), etc. If the parent cyanobacterium belongs to the genus Cynahothece, it may be csaB (NCBI access ID: WP_107667006.1), etc., and if the parent cyanobacterium belongs to the genus Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc. , if the parent cyanobacteria belongs to the genus Calothrix, it may be csaB (NCBI access ID: WP_096658142.1), etc., and if the parent cyanobacteria belongs to the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc. If the parent cyanobacterium belongs to the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), and if the parent cyanobacterium belongs to the genus Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735). etc. may be used. The base sequences of these genes can be obtained from the NCBI database or Cyanobase mentioned above.

More specifically, the gene encoding cell wall-pyruvate modification enzyme 9 is slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC 6803, synpcc7942_1529 (SEQ ID NO: 11) of Synechococcus sp. PCC 7942, or Anabaena cylindrica PCC. Anacy_1623 (SEQ ID NO: 12) of 7122 may be used. Alternatively, the gene may have a base sequence that is 50% or more identical to these genes.

As a result, in the modified cyanobacteria, the base sequence of the gene encoding any of the cell wall-pyruvic acid modifying enzymes 9 shown in SEQ ID NOs: 10 to 12 above or the gene encoding any of these enzymes is 50% or more. Genes that are identical are deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above cell wall-pyruvate modifying enzymes 9 or a protein having a function equivalent to any of these enzymes is suppressed, or (ii) the above-mentioned The function of any cell wall-pyruvate modifying enzyme 9 or a protein having a function equivalent to any of these enzymes is suppressed. This makes it difficult for the covalent sugar chains 3 on the surface of the cell wall 4 to be modified with pyruvate, so that the sugar chains 3 on the cell wall 4 interact with the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding strength are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the amount of modification of sugar chains 3 with pyruvate for bonding the cell wall 4 to the outer membrane 5 is reduced, so that the binding strength between the cell wall 4 and the outer membrane 5 is reduced. is weakened, and the outer membrane 5 becomes easily partially detached from the cell wall 4.

As mentioned above, if the base sequences of genes encoding proteins are 30% or more identical, it is considered that there is a high possibility that a protein having the same function as the protein will be expressed. Therefore, as a gene encoding cell wall-pyruvate modifying enzyme 9 whose function is suppressed, for example, any of the base sequences of the genes encoding cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs: 10 to 12 above. and a base sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, still more preferably 90% or more, In addition, it may be a gene encoding a protein or polypeptide that has the function of catalyzing a reaction that modifies the covalent sugar chain 3 of the peptidoglycan 2 of the cell wall 4 with pyruvate.

[6. Method for producing modified cyanobacteria]
Next, a method for producing modified cyanobacteria according to this embodiment will be explained. The method for producing a modified cyanobacterium includes the step of suppressing the function of a protein involved in bonding the outer membrane 5 and the cell wall 4 in the cyanobacterium.

In the present embodiment, the protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvic acid modifying enzyme 9.

Means for suppressing protein function include, but are not particularly limited to, deletion or inactivation of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. It may be possible to inhibit the transcription of these genes, inhibit the translation of the transcripts of these genes, or administer an inhibitor that specifically inhibits these proteins.

Means for deleting or inactivating the above gene include, for example, introducing a mutation into one or more bases on the base sequence of the gene, replacing the base sequence with another base sequence, or replacing the base sequence with another base sequence. It may be insertion, or deletion of part or all of the base sequence of the gene.

Means for inhibiting the transcription of the above genes include, for example, introducing mutations into the promoter region of the gene, inactivating the promoter by substituting or inserting other base sequences, or CRISPR interference (non-transfer). Patent Document 13: Yao et al., ACS Synth. Biol., 2016, 5:207-212). Specific methods for the above-mentioned mutagenesis or base sequence substitution or insertion may be, for example, ultraviolet irradiation, site-specific mutagenesis, or homologous recombination.

Further, the means for inhibiting the translation of the transcription product of the gene may be, for example, RNA (ribonucleic acid) interference.

By using any of the above means, the function of a protein involved in binding the outer membrane 5 and cell wall 4 in cyanobacteria may be suppressed to produce a modified cyanobacterium.

As a result, in the modified cyanobacteria produced by this production method, the bond between the cell wall 4 and the outer membrane 5 (that is, the amount of bond and binding force) is partially reduced, so that the outer membrane 5 is partially separated from the cell wall 4. It becomes easier to detach. Therefore, in the modified cyanobacteria, proteins produced within the bacterial cells tend to leak out of the outer membrane 5 (that is, out of the bacterial cells). Therefore, according to the method for producing modified cyanobacteria according to the present embodiment, modified cyanobacteria with improved protein secretion productivity can be provided.

Furthermore, in the modified cyanobacteria produced by this production method, the proteins produced within the bacterial cells leak out of the bacterial cells, so there is no need to disrupt the bacterial cells to recover the proteins. For example, it is sufficient to culture the modified cyanobacteria under appropriate conditions and then collect the proteins secreted into the culture solution, so it is also possible to collect the proteins in the culture solution while culturing the modified cyanobacteria. Therefore, by using the modified cyanobacteria obtained by this production method, efficient microbial protein production can be carried out. Therefore, according to the method for producing a modified cyanobacterium according to the present embodiment, it is possible to provide a modified cyanobacterium with high utilization efficiency that can be used repeatedly even after protein recovery.

Note that the modified cyanobacteria produced by this production method secretes, to the outside of the cell, a group of proteins that are originally present in the periplasm, such as peptidases or phosphatases. In this embodiment, for example, by modifying a gene encoding a protein originally produced within cyanobacterial cells, such as a group of proteins present in the periplasm, and replacing it with a gene encoding another protein, It is also possible to cause modified cyanobacteria to produce desired proteins. Therefore, according to the method for producing a modified cyanobacterium according to the present embodiment, it is also possible to provide a modified cyanobacterium that can easily and efficiently produce a desired protein.

[7. Protein production method using modified cyanobacteria]
Next, a method for producing proteins using the modified cyanobacteria according to the present embodiment will be explained. The method for producing proteins using modified cyanobacteria according to the present embodiment includes the step of culturing the modified cyanobacteria described above.

Cultivation of cyanobacteria can generally be carried out based on liquid culture using BG-11 medium (see Table 2) or a modified method thereof. Therefore, culturing of modified cyanobacteria may be carried out in the same manner. In addition, the culture period of cyanobacteria for protein production may be any period that allows the cells to grow sufficiently and the protein to accumulate at a high concentration, for example, 1 to 3 days. It may be for 4 to 7 days. Further, the culture method may be, for example, aeration agitation culture or shaking culture.

By culturing under the above conditions, the modified cyanobacteria produce proteins within their cells and secrete the proteins into the culture solution. When recovering proteins secreted into a culture solution, solids such as cells (so-called bacterial bodies) are removed from the culture solution by filtration or centrifugation, and the culture supernatant is recovered. You can. According to the method for producing proteins using modified cyanobacteria according to the present embodiment, since proteins are secreted outside the cells of modified cyanobacteria, there is no need to disrupt cells for protein recovery. Therefore, the modified cyanobacteria remaining after protein recovery can be used repeatedly to produce proteins.

Note that the method for recovering proteins secreted into the culture solution is not limited to the above example, and proteins in the culture solution may be recovered while culturing the modified cyanobacteria. For example, by using a permeable membrane that allows proteins to permeate, the protein that has passed through the permeable membrane may be recovered. In this case, useful microorganisms such as lactic acid bacteria may be cultured using the protein that has passed through the permeable membrane as a nutrient source. In this way, proteins in the culture solution can be recovered while culturing the modified cyanobacteria, so that there is no need to remove the cells of the modified cyanobacteria from the culture solution. Therefore, proteins can be produced more easily and efficiently.

Moreover, since the recovery process of bacterial cells from the culture solution and the process of crushing the bacterial cells are not necessary, damage and stress to the modified cyanobacteria can be reduced. Therefore, the protein secretion productivity of the modified cyanobacteria is less likely to decrease, and the modified cyanobacteria can be used for a longer period of time.

As described above, according to the method for producing proteins using modified cyanobacteria according to the present embodiment, enzymes for producing food ingredient raw materials or compounds, diagnostic enzymes or therapeutic enzymes in the medical field, or agricultural, agricultural, and agricultural Enzymes for feed in the livestock industry can be easily and efficiently obtained.

[Example]
Hereinafter, a method for identifying outer membrane detachment of cyanobacteria and a device for identifying outer membrane detachment of cyanobacteria of the present disclosure will be specifically explained in Examples, but the present disclosure is in no way limited to the following Examples. It's not something you can do.

In the following examples, as a method for partially detaching the outer membrane of cyanobacteria from the cell wall, we will suppress the expression of the slr1841 gene encoding an SLH domain-retaining outer membrane protein (Example 1) and modify the cell wall with pyruvate. The expression of the slr0688 gene encoding the enzyme was suppressed (Example 2), and two types of modified cyanobacteria were produced. Then, we measured the protein secretion productivity of these modified cyanobacteria and identified the secreted proteins. The cyanobacterial species used in this example is Synechocystis sp. PCC 6803 (hereinafter simply referred to as "cyanobacteria").

(Example 1)
In Example 1, a modified cyanobacterium in which the expression of the slr1841 gene encoding the SLH domain-retaining outer membrane protein was suppressed was produced.

(1) Construction of a modified cyanobacterial strain in which slr1841 gene expression is suppressed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference method was used as a method for suppressing gene expression. In this method, the expression of the slr1841 gene is suppressed by introducing the gene encoding the dCas9 protein (hereinafter referred to as the dCas9 gene) and the slr1841_sgRNA (single-guide Ribonucleic Acid) gene into the chromosomal DNA of cyanobacteria. Can be done. Furthermore, by controlling the transcriptional activity of slr1841_sgRNA, the degree of suppression of the slr1841 gene can be controlled.

The mechanism of gene expression suppression using this method is as follows.

First, a Cas9 protein lacking nuclease activity (dCas9) and sgRNA (slr1841_sgRNA) that binds complementary to the base sequence of the slr1841 gene form a complex.

Next, this complex recognizes the slr1841 gene on the cyanobacterial chromosomal DNA and specifically binds to the slr1841 gene. This binding causes steric hindrance, which inhibits transcription of the slr1841 gene. As a result, the expression of the cyanobacterial slr1841 gene is suppressed. Furthermore, by controlling the transcriptional activity of slr1841_sgRNA, the degree of suppression of the slr1841 gene can be controlled.

Hereinafter, a method for introducing each of the above two genes into the chromosomal DNA of cyanobacteria will be specifically explained.

(1-1) Introduction of dCas9 gene Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (see Non-Patent Document 13) as a template, the dCas9 gene and an operator gene for controlling the expression of the dCas9 gene, and The spectinomycin resistance marker gene, which serves as a marker for gene introduction, was amplified by the PCR (polymerase chain reaction) method using the primers psbA1-Fw (SEQ ID NO: 13) and psbA1-Rv (SEQ ID NO: 14) listed in Table 1. . In the LY07 strain, the three genes mentioned above are inserted into the psbA1 gene on the chromosomal DNA in a linked state, so they can be amplified as one DNA fragment by PCR. Here, the obtained DNA fragment is referred to as "psbA1::dCas9 cassette." The psbA1::dCas9 cassette was inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark) to obtain the pUC19-dCas9 plasmid.

1 μg of the obtained pUC19-dCas9 plasmid was mixed with a cyanobacterial culture solution (bacterial cell concentration OD730 = approximately 0.5), and the pUC19-dCas9 plasmid was introduced into cyanobacterial cells by natural transformation. Transformed cells were selected by growing on BG-11 agar medium containing 20 μg/mL spectinomycin. In the selected cells, homologous recombination occurs between the psbA1 gene on the chromosomal DNA and the psbA1 upstream fragment region and psbA1 downstream fragment region on the pUC19-dCas9 plasmid. As a result, a Synechocystis dCas9 strain in which the dCas9 cassette was inserted into the psbA1 gene region was obtained. The composition of the BG-11 medium used is shown in Table 2.

(1-2) Introduction of slr1841_sgRNA gene In CRISPR interference, sgRNA specifically binds to the target gene by introducing a sequence of approximately 20 bases complementary to the target sequence into a region called protospacer on the sgRNA gene. do. The protospacer sequences used in this example are shown in Table 3.

In Synechocystis LY04, LY05, or LY07 strains, the sgRNA gene (excluding the protospacer region) and the kanamycin resistance marker gene are inserted into the slr2030-slr2031 genes on the chromosomal DNA (Non-patent Document (see 13). Therefore, by adding a protospacer sequence (SEQ ID NO: 21) complementary to the slr1841 gene (SEQ ID NO: 7) to the primers used when amplifying the sgRNA gene by PCR method, we created an sgRNA (slr1841_sgRNA) that specifically recognizes slr1841. ) can be easily obtained. Furthermore, by controlling the transcriptional activity of slr1841_sgRNA, the degree of suppression of the slr1841 gene can be controlled.

First, using the chromosomal DNA of the LY07 strain as a template, a set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr1841-Rv (SEQ ID NO: 16) listed in Table 1, as well as sgRNA_slr1841-Fw (SEQ ID NO: 17) and slr2031- Two DNA fragments were amplified by PCR using a set of Rv (SEQ ID NO: 18).

Next, by using the mixed solution of the above DNA fragments as a template and using the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1 by PCR method, ( A DNA fragment (slr2030-2031::slr1841_sgRNA) was obtained in which i) slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) kanamycin resistance marker gene, and (iv) slr2031 gene fragment were linked in this order. Using the In-Fusion PCR cloning method (registered trademark), slr2030-2031::slr1841_sgRNA was inserted into the pUC19 plasmid to obtain the pUC19-slr1841_sgRNA plasmid.

The pUC19-slr1841_sgRNA plasmid was introduced into the Synechocystis dCas9 strain in the same manner as in (1-1) above, and the transformed cells were selected on a BG-11 agar medium containing 30 μg/mL kanamycin. As a result, a transformant Synechocystis dCas9 slr1841_sgRNA strain (hereinafter also referred to as slr1841 suppressed strain) in which slr1841_sgRNA was inserted into the slr2030-slr2031 gene on the chromosomal DNA was obtained.

(1-3) Suppression of slr1841 gene The promoter sequences of the above dCas9 gene and slr1841_sgRNA gene are designed so that their expression is induced in the presence of anhydrotetracycline (aTc). In this example, expression of the slr1841 gene was suppressed by adding aTc at a final concentration of 1 μg/mL to the medium.

As described above, in Example 1, the total amount of proteins involved in bonding the outer membrane and cell wall in cyanobacteria was adjusted to the parent strain (Synechocystis dCas9 strain, Comparative Example 1 described below) without impairing the cell growth ability. We obtained a modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain (so-called slr1841 suppressed strain) in which the amount of the protein was suppressed to about 30% compared to the amount of the protein in . Here, the proteins involved in the binding between the outer membrane and the cell wall are slr1841, slr1908, and slr0042.

(Example 2)
In Example 2, a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modifying enzyme was suppressed was obtained by the following procedure.

(2) Construction of a modified cyanobacterial strain in which the expression of the slr0688 gene is suppressed By the same procedure as in (1-2) above, sgRNA containing the protospacer sequence (SEQ ID NO: 22) complementary to the slr0688 gene (SEQ ID NO: 4) The gene was introduced into Synechocystis dCas9 strain to obtain Synechocystis dCas9 slr0688_sgRNA strain. In addition, the set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr0688-Rv (SEQ ID NO: 19) and the set of sgRNA_slr0688-Fw (SEQ ID NO: 20) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1 were used. In-Fusion PCR was performed on a DNA fragment (slr2030-2031::slr0688_sgRNA) in which (i) slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) kanamycin resistance marker gene, and (iv) slr2031 gene fragment were linked in this order. The procedure was carried out under the same conditions as in (1-2) above, except that the cloning method (registered trademark) was used to insert into the pUC19 plasmid to obtain the pUC19-slr0688_sgRNA plasmid. Furthermore, by controlling the transcriptional activity of slr0688_sgRNA, the degree of suppression of the slr0688 gene can be controlled.

Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in (1-3) above.

As described above, in Example 2, the amount of proteins involved in bonding the outer membrane and cell wall in cyanobacteria was determined from the parent strain (Synechocystis dCas9 strain, Comparative Example 1 described below) without impairing the cell growth ability. ), a modified cyanobacterial Synechocystis dCas9 slr0688_sgRNA strain (hereinafter also referred to as slr0688 suppressed strain) was obtained, in which the amount of the protein was suppressed to about 50%. Here, the protein involved in binding the outer membrane and the cell wall is slr0688.

(Comparative example 1)
In Comparative Example 1, Synechocystis dCas9 strain was obtained by the same procedure as in Example 1 (1-1).

Subsequently, the cell surface conditions of the bacterial strains obtained in Example 1, Example 2, and Comparative Example 1 were observed and protein secretion productivity tests were conducted. The details will be explained below.

(3) Observation of cell surface state of bacterial strains The modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (so-called slr1841 suppressed strain), the modified cyanobacterium Synechocystis dCas9 slr0688_sgRNA strain obtained in Example 2 (so-called, slr0688 suppressed strain) and the modified cyanobacterium Synechocystis dCas9 strain obtained in Comparative Example 1 (hereinafter referred to as Control strain), ultrathin sections were prepared, and the state of the cell surface layer (in other words, Adventitial structure) was observed.

(3-1) Culture of the strain The slr1841 suppressed strain from Example 1 was inoculated into 12 mL of BG-11 medium (see Table 2) containing 1 μg/mL aTc so that the initial cell concentration was OD730 = 0.05. The cells were cultured with shaking for 5 days at a light intensity of 100 μmol/m ² /s and a temperature of 30°C. Note that the slr0688 suppressed strain of Example 2 and the Control strain of Comparative Example 1 were also cultured under the same conditions as in Example 1.

(3-2) Preparation of ultrathin sections of the bacterial strain The culture solution obtained in (3-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to collect cells of the slr1841 suppressed strain of Example 1. . Cells were then quickly frozen in liquid propane at -175°C, and then fixed at -80°C for 2 days using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, the dehydrated cells were permeated with propylene oxide, and then submerged in a resin (Quetol-651) solution. Thereafter, the resin was left to stand at 60°C for 48 hours to harden the resin, and the cells were embedded in the resin. The cells in the resin were sliced into 70 nm thick sections using an ultramicrotome (Ultracut) to create ultrathin sections. This ultrathin section was stained with a 2% uranium acetate and 1% lead citrate solution to prepare a transmission electron microscopy sample of the slr1841 suppressed strain of Example 1. Note that the same operation was performed for the slr0688 suppressed strain of Example 2 and the Control strain of Comparative Example 1, respectively, to prepare samples for transmission electron microscopy.

(3-3) Observation using an electron microscope The ultrathin section obtained in (3-2) above was observed using a transmission electron microscope (JEOL JEM-1400Plus) at an accelerating voltage of 100 kV. The observation results are shown in FIGS. 4 to 9.

First, the slr1841 suppressed strain of Example 1 will be explained. FIG. 4 is a TEM (Transmission Electron Microscope) image of the slr1841 suppressed strain of Example 1. FIG. 5 is an enlarged image of the broken line area A in FIG. 5(a) is an enlarged TEM image of the broken line area A in FIG. 4, and FIG. 5(b) is a diagram depicting the enlarged TEM image of FIG. 5(a).

As shown in Figure 3, in the slr1841 suppressed strain of Example 1, the outer membrane was partially peeled off from the cell wall (that is, the outer membrane was partially peeled off), and the outer membrane was partially bent. there was.

In order to confirm the condition of the cell surface layer in more detail, we enlarged the area A shown by the broken line and found that the outer membrane had partially peeled off, as shown in Figures 5(a) and 5(b). The parts (dot-dashed line areas a1 and a2 in the figure) could be confirmed. Further, a portion where the adventitia was largely bent was confirmed near the dotted line area a1. This region is a region where the bond between the outer membrane and the cell wall is weakened, and it is thought that the culture solution permeated from the outer membrane into the periplasm, causing the outer membrane to swell outward and become bent.

Next, the slr0688 suppressed strain of Example 2 will be explained. FIG. 6 is a TEM image of the slr0688 suppressed strain of Example 2. FIG. 7 is an enlarged image of the broken line area B in FIG. 7(a) is an enlarged TEM image of the broken line area B in FIG. 6, and FIG. 7(b) is a diagram depicting the enlarged TEM image of FIG. 7(a).

As shown in FIG. 6, in the slr0688-inhibited strain of Example 2, the outer membrane was partially detached from the cell wall, and the outer membrane was partially bent. Furthermore, in the slr0688 suppressed strain, it was confirmed that the outer membrane was partially detached from the cell wall.

In order to confirm the state of the cell surface layer in more detail, we enlarged and observed the broken line area B. As shown in FIGS. The dotted line area b1 in the figure and the parts where the outer membrane had partially peeled off (dot and dot line areas b2 and b3 in the figure) were confirmed. In addition, parts where the outer membrane was detached from the cell wall were confirmed near each of the dotted line areas b1, b2, and b3.

Next, the Control strain of Comparative Example 1 will be explained. FIG. 8 is a TEM image of the Control strain of Comparative Example 1. FIG. 9 is an enlarged image of the broken line area C in FIG. 9(a) is an enlarged TEM image of the broken line area C in FIG. 8, and FIG. 9(b) is a diagram depicting the enlarged TEM image of FIG. 9(a).

As shown in FIGS. 8 and 9, the cell surface layer of the Control strain of Comparative Example 1 was well-organized, and the inner membrane, cell wall, outer membrane, and S layer remained laminated in this order. In other words, in the Control strain, as in Examples 1 and 2, the parts where the outer membrane detached from the cell wall, the parts where the outer membrane peeled off from the cell wall (that is, the parts fell off), and the parts where the outer membrane bent were I couldn't see it.

(4) Protein secretion productivity test The slr1841 suppressed strain of Example 1, the slr0688 suppressed strain of Example 2, and the Control strain of Comparative Example 1 were cultured, and the amount of protein secreted outside the cells (hereinafter referred to as secreted (also referred to as protein amount) was measured. The protein secretion productivity of each of the above bacterial strains was evaluated based on the amount of protein in the culture solution. Note that protein secretion productivity refers to the ability to produce proteins by secreting proteins produced within the cells to the outside of the cells. A specific method will be explained below.

(4-1) Culture of strain The slr1841 suppressed strain of Example 1 was cultured in the same manner as in (3-1) above. Culturing was performed independently three times. The strains of Example 2 and Comparative Example 1 were also cultured under the same conditions as the strains of Example 1.

(4-2) Quantification of extracellularly secreted proteins The culture solution obtained in (4-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to obtain a culture supernatant. The obtained culture supernatant was filtered using a membrane filter with a pore size of 0.22 μm to completely remove the cells of the slr1841 suppressed strain of Example 1. The total amount of protein contained in the culture supernatant after filtration was quantified by the BCA (Bicinchoninic acid) method. This series of operations was performed for each of the three independently cultured cultures, and the average value and standard deviation of the amount of protein secreted extracellularly of the slr1841 suppressed strain of Example 1 was determined. In addition, for the strains of Example 2 and Comparative Example 1, the protein in the three culture solutions was quantified under the same conditions, and the average value and standard deviation of the protein amounts in the three culture solutions were determined.

The results are shown in Figure 10. FIG. 10 is a graph showing the amount of protein in the culture supernatants of the modified cyanobacteria of Example 1, Example 2, and Comparative Example 1 (n=3, error bar=SD).

As shown in FIG. 10, both the slr1841 suppressed strain of Example 1 and the slr0688 suppressed strain of Example 2 secreted more protein (mg/ L) was improved by about 25 times.

Although the data are omitted, the absorbance (730 nm) of the culture solution was measured and the amount of secreted protein per 1 g of bacterial cell dry weight (mg protein/g cell dry weight) was calculated. In both of the slr0688 suppressed strain of Example 2, the amount of secreted protein per gram of bacterial cell dry weight (mg protein/g cell dry weight) was approximately 36 times higher than that of the Control strain of Comparative Example 1. Ta.

Furthermore, as shown in FIG. 10, the gene encoding the cell wall-pyruvate modifying enzyme ( The slr0688 suppressed strain of Example 2, in which the expression of slr0688) was suppressed, had a higher amount of protein secreted into the culture supernatant. This is thought to be related to the fact that the number of covalently bonded sugar chains on the cell wall surface is greater than the number of SLH domain-retaining outer membrane proteins (Slr1841) in the outer membrane. In other words, in the slr0688 suppressed strain of Example 2, the binding amount and binding strength between the outer membrane and the cell wall were lower than in the slr1841 suppressed strain of Example 1, so the amount of secreted protein was lower than that of the slr1841 suppressed strain of Example 1. It is thought that the amount exceeded that of stocks.

From the above results, by suppressing the function of proteins related to the bond between the outer membrane and the cell wall, the bond between the outer membrane and the cell wall of cyanobacteria is partially weakened, and the outer membrane partially detaches from the cell wall. I was able to confirm that it was released. It was also confirmed that by weakening the bond between the outer membrane and the cell wall, proteins produced within the cyanobacterial cell can more easily leak out of the cell. Therefore, it was shown that the modified cyanobacteria and the method for producing the same according to the present embodiment can provide modified cyanobacteria with greatly improved protein secretion productivity.

(5) Identification of secreted proteins Next, secreted proteins contained in the culture supernatant obtained in (4-2) above were identified by LC-MS/MS. The method will be explained below.

(5-1) Sample preparation Add 8 times the amount of cold acetone to the culture supernatant, leave it at 20°C for 2 hours, and centrifuge at 20,000 g for 15 minutes to obtain a protein precipitate. . 100 mM Tris pH 8.5, 0.5% sodium dodecanoate (SDoD) was added to this precipitate, and the protein was dissolved using a closed ultrasonicator. After adjusting the protein concentration to 1 μg/mL, dithiothreitol (DTT) with a final concentration of 10 mM was added and left at 50°C for 30 minutes. Subsequently, iodoacetamide (IAA) was added at a final concentration of 30 mM, and the mixture was allowed to stand at room temperature (protected from light) for 30 minutes. To stop the IAA reaction, cysteine was added at a final concentration of 60 mM, and the mixture was allowed to stand at room temperature for 10 minutes. 400 ng of trypsin was added and left standing at 37°C overnight to fragment the protein into peptide fragments. After adding 5% TFA (Trifluoroacetic Acid), the mixture was centrifuged at 15,000 g for 10 minutes at room temperature to obtain a supernatant. This work removed SDoD. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. Thereafter, 3% acetonitrile and 0.1% formic acid were added, and the sample was dissolved using a closed ultrasonic crusher. The peptide concentration was adjusted to 200 ng/μL.

(5-2) LC-MS/MS Analysis The sample obtained in (5-1) above was analyzed using an LC-MS/MS device (UltiMate 3000 RSLCnano LC System) under the following conditions.

Sample injection amount: 200 ng
Column: CAPCELL CORE MP 75 μm × 250 mm
Solvent: A solvent is 0.1% formic acid aqueous solution, B solvent is 0.1% formic acid + 80% acetonitrile Gradient program: B solvent 8% after 4 minutes of sample injection, B solvent 44% after 27 minutes, B solvent 80% after 28 minutes, 34 Measurement ended after 5-3 minutes. (5-3) Data analysis The obtained data was analyzed under the following conditions, and proteins and peptides were identified and quantitative values were calculated.

Software: Scaffold DIA
Database: UniProtKB/Swiss Prot database ( Synechocystis sp. PCC 6803)
Fragmentation: HCD
Precursor Tolerance: 8 ppm
Fragment Tolerance: 10 ppm
Data Acquisition Type: Overlapping DIA
Peptide Length: 8-70
Peptide Charge: 2-8
Max Missed Cleavages: 1
Fixed Modification: Carbamidomethylation
Peptide FDR: 1% or less Among the identified proteins, 10 types of proteins are shown in Table 4 in order of the highest relative quantitative value.

All 10 types of proteins were contained in the culture supernatants of the slr1841 suppressed strain of Example 1 and the slr0688 suppressed strain of Example 2, respectively. All of these proteins retained periplasmic (referring to the space between the outer and inner membrane) translocation signals. This result shows that in the modified strains of Examples 1 and 2, the outer membrane partially detaches from the cell wall, making it easier for proteins in the periplasm to leak out of the outer membrane (i.e., outside the bacterial cell). This was confirmed. Therefore, it was shown that the modified cyanobacterium according to the present embodiment has significantly improved protein secretion productivity.

(6) Determination of outer membrane detachment of cyanobacteria (6-1) Cultivation of cyanobacteria Using the Control strain of Comparative Example 1 as a wild strain of cyanobacterium Synechocystis sp. PCC 6803, outer membrane detachment type Synechocystis sp. PCC 6803 was used. As a mutant strain, the slr0688 suppressed strain of Example 2, which was confirmed to have exfoliated outer membrane by the protein secretion production test described in (4) above, was used. Hereinafter, these cyanobacteria will be referred to as cyanobacteria wild strain and outer membrane-exfoliating cyanobacteria, respectively. These two types of cyanobacteria were inoculated into 12 mL of BG-11 medium (see Table 2) containing 1 μg/mL aTc in a 125 mL Erlenmeyer flask at an initial bacterial concentration OD730 = 0.1. The cells were cultured with shaking for 3 days at a light intensity of 100 μmol/m ² /s and at 30°C.

After 3 days of culture, the culture solution was centrifuged at 2,500 g for 10 minutes at room temperature, and the culture supernatant was filtered through Millex-GV Syringe Filter Unit, 0.22 μm (Millipore). This filtered supernatant was subjected to amino acid analysis by LC-MS/MS.

(6-2) Amino acid analysis by LC-MS/MS Next, isoleucine, leucine, tyrosine, and phenylalanine contained in the culture supernatant of the two types of cyanobacteria obtained in (6-1) above were analyzed. Measured by LC-MS/MS. The method will be explained below.

(6-2-1) Sample preparation After lyophilizing 1 mL of the culture supernatant of the cyanobacterial wild strain obtained in (6-1) above, it was added to 195 μL of borate buffer for APDS tag (registered trademark) Wako. Dissolved. Add 5 μL of a solution of 100 mg of APDS (3-aminopyridyl-N-hydroxysuccinimidyl carbamate) tag reagent dissolved in 5 mL acetonitrile to this, mix well, and incubate at 55-60℃ for 5-15 minutes. Heated. The culture supernatant of outer membrane-exfoliating cyanobacteria was also treated in the same manner.

(6-2-2) LC-MS/MS analysis The sample obtained in (6-2-1) above was analyzed using an LC-MS/MS device (UltiMate 3000 RSLCnano LC System) under the following conditions. did.

Sample injection volume: 10 μL
Column: Tosoh TSKgel ODS-100V 2.0mmI.D×15cm
Mobile phase: Solution A is APDS tag (registered trademark) eluent for Wako, solution B is 60% acetonitrile
Elution timetable (B solution %):
0 min, 6%; 0.05-1.70 min, 8%; 1.71 min, 12%; 4.95 min, 30%; 5.95 min, 60%; 6.70 min, 95%; 6.71 min, 6%; 12.0 min, 6%
Flow rate: 0.3 mL/min
Column temperature: 40℃
Ionization mode: ESI positive
(6-3) Results FIG. 11 is a diagram showing the results of amino acid analysis by LC-MS/MS of culture supernatants of two types of cyanobacteria. Figure 11 shows the TIC (total ion chromatogram) of the culture supernatant of outer membrane exfoliating cyanobacteria and the culture supernatant of cyanobacteria wild strain, where A is the peak derived from tyrosine and B is the peak derived from isoleucine. C is a peak derived from leucine, and D is a peak derived from phenylalanine. As shown in Figure 11, the peaks A to D are almost not detected in the culture supernatant of wild-type cyanobacteria, but are detected in the culture supernatant of outer membrane-exfoliating cyanobacteria. When cyanobacteria that had detached from the cell wall were cultured, it was confirmed that they were characteristically secreted into the culture supernatant.

Table 5 also shows the measurement results of the concentrations of the four amino acids mentioned above. As shown in Table 5, it was confirmed that isoleucine, leucine, tyrosine, and phenylalanine were present in the culture supernatant of outer membrane-exfoliating cyanobacteria at a concentration of approximately 130 nM or more and 320 nM or less. On the other hand, it was confirmed that these four amino acids were hardly present in the culture supernatant of the cyanobacterial wild strain.

Therefore, it was confirmed that by quantifying at least one amino acid selected from the group consisting of these four types of amino acids in the culture supernatant, it is possible to easily determine whether the outer membrane has detached from the cell wall. . From the results in Table 5, it can be considered that if the concentration of each amino acid exceeds 100 nM, it may be determined that the outer membrane of the cyanobacterium is detached from the cell wall. By applying the method for determining outer membrane detachment of cyanobacteria of the present disclosure, there is no need to collect cyanobacterial cells and confirm the state of the outer membrane using complicated methods such as electron microscopy, which is quick and easy. In addition, it becomes possible to select cells in a state suitable for substance production and apply them to substance production.

(7) Discussion In this example, it was confirmed that the modified cyanobacteria of the present disclosure secretes proteins present inside the bacterial cells (in the periplasm in this case) to the outside of the bacterial cells. The modified cyanobacteria of the present disclosure can, for example, be genetically modified to produce other proteins instead of the proteins identified above (i.e., proteins originally produced within the bacterial body), Desired proteins can be efficiently produced. In addition, cyanobacteria have a high photosynthetic ability, so by culturing them with light, water, air, and small amounts of inorganic substances, they can easily obtain the necessary proteins when needed. There is no need to use complicated equipment. Furthermore, proteins tend to lose their functions when processed into, for example, supplements. Therefore, according to the modified cyanobacteria of the present disclosure, it is possible to provide proteins while maintaining their functions. Due to the above advantages, the modified cyanobacteria of the present disclosure are expected to be applied in various fields.

In addition, in "(3-3) Observation using an electron microscope," it was found that in modified strains in which the expression of genes encoding proteins related to the bond between the outer membrane and the cell wall was suppressed, the bond between the outer membrane and the cell wall was weakened. It was confirmed that the outer membrane had partially peeled off and that the outer membrane had partially expanded. This is due to the weakening of the bond between the outer membrane and the cell wall, which makes it easier for culture fluid to flow in from outside the cell due to osmotic pressure to areas where the outer membrane has partially detached from the cell wall, and the inflowing culture fluid causes the outer membrane to is thought to have partially expanded. Since there is a large difference between the protein concentration of the culture solution and the protein concentration in the periplasm, the culture solution flowing from the outer membrane into the periplasm never stops, and the outer membrane, which can no longer withstand the internal pressure, ruptures and peels off. It is thought that it has fallen off (in other words, it has peeled off).

In addition, in Table 3, among all the proteins identified by LC-MS/MS analysis, only 10 proteins are listed in order of the highest relative quantitative value. In "(5-3) Data analysis," all 10 types of proteins listed in Table 3 were found to be contained in the culture supernatants of the slr1841 suppressed strain in Example 1 and the slr0688 strain in Example 2. Although described, all other proteins identified by LC-MS/MS analysis were similarly confirmed to be contained in each of the culture supernatants of Example 1 and Example 2. It was confirmed that the secreted proteins include proteins that originally exist in the periplasm and proteins that are transported from the cytoplasm to the periplasm and function within the periplasm.

Table 5 also lists the concentrations of four amino acids characteristically contained in the culture supernatant of outer membrane-exfoliating cyanobacteria as determined by LC-MS/MS analysis. These four amino acids were hardly present in the culture supernatant of the cyanobacterial wild strain. Therefore, it was confirmed that if the concentration of any of these four amino acids in the culture supernatant exceeds a threshold value (e.g., 100 nM), it can be determined that the outer membrane of cyanobacteria has detached from the cell wall. Ta.

(Other embodiments)
Above, the method for determining outer membrane detachment of cyanobacteria, the apparatus for determining outer membrane detachment of cyanobacteria, and the program according to the present disclosure have been described based on the embodiments, but the present disclosure is limited to these embodiments. It is not something that will be done. Unless departing from the spirit of the present disclosure, various modifications to the embodiments that can be thought of by those skilled in the art and other forms constructed by combining some of the constituent elements of the embodiments are also within the scope of the present disclosure. included.

In the above embodiment, the total amount of proteins involved in the binding between the outer membrane and the cell wall in cyanobacteria is suppressed to 30% or more and 70% or less of the total amount of the protein in the parent strain, so that the outer membrane and the cell wall are bonded together. Although an example has been described in which the binding is weakened and a protein produced within the bacterial cell leaks out of the bacterial cell, the present disclosure is not limited thereto. For example, by applying an external force to cyanobacteria, the bond between the outer membrane and the cell wall may be weakened, or the outer membrane may be weakened. Alternatively, the outer membrane may be weakened by adding an enzyme or a drug to the culture solution of cyanobacteria.

According to the present disclosure, it is possible to easily determine whether or not the outer membrane of cyanobacteria has peeled off from the cell wall, so it is possible to easily determine whether the cyanobacteria are in a state suitable for substance production, and whether the cyanobacteria are in a state suitable for substance production even during the culture process. It is possible to quickly determine whether or not a suitable state is being maintained. Therefore, according to the present disclosure, it is possible to select and use cyanobacteria in a state suitable for material production, thereby making it possible to reliably improve material productivity.

1 Inner membrane 2 Peptidoglycan 3 Sugar chain 4 Cell wall 5 Outer membrane 6 SLH domain-retaining outer membrane protein 7 SLH domain 8 Organic channel protein 9 Cell wall-pyruvate modifying enzyme

Claims

a measuring step of measuring the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria;
a determining step of determining whether the outer membrane of the cyanobacterium has peeled off from the cell wall, based on the concentration of the at least one amino acid measured in the measuring step;
including,
Method for determining outer membrane detachment of cyanobacteria.
In the measurement step, the concentration of four amino acids, isoleucine, leucine, tyrosine, and phenylalanine, is measured,
In the determination step, it is determined whether the outer membrane of the cyanobacterium has peeled off from the cell wall, based on the concentrations of the four amino acids.
The method for determining outer membrane detachment of cyanobacteria according to claim 1.
In the determination step, if at least one of the concentrations of the at least one amino acid is equal to or higher than a threshold value, it is determined that the outer membrane of the cyanobacterium is detached from the cell wall, and all of the concentrations of the at least one amino acid are determined to be detached from the cell wall. is below the threshold, determining that the outer membrane of the cyanobacterium has not peeled off from the cell wall;
The method for determining outer membrane detachment of cyanobacteria according to claim 1.
the threshold value is 100 nM;
The method for determining outer membrane detachment of cyanobacteria according to claim 3.
a measurement unit that measures the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria;
a determining unit that determines whether the outer membrane of the cyanobacterium has peeled off from the cell wall, based on the concentration of the at least one amino acid measured by the measuring unit;
Equipped with
Device for determining outer membrane detachment of cyanobacteria.
Based on the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of the cyanobacteria, determining whether the outer membrane of the cyanobacteria is detached from the cell wall. to make the computer execute the method
program.
A method for determining outer membrane detachment of cyanobacteria, comprising a determination step of determining whether or not the outer membrane of the cyanobacteria has detached from the cell wall, based on the concentration of amino acids in the culture supernatant of the cyanobacteria. ,
The amino acid is at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine.
Method for determining outer membrane detachment of cyanobacteria.